Role of the ubiquitin-like modifier FAT10 in protein degradation and ...

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inding binding accelerated to to degradation FAT10 proteasome of FAT10 NUB1L + + + NUB1∆UBA1-3 - + + NUB1∆UBL + - - FAT10-GFP + + + FAT10-N-GFP - + + FAT10-C-GFP + + + Chapter 1 Table 1: A summary of the results obtained. Results are classified with (+) for a positive result in a binding assay or for accelerated degradation and with (-) for a negative result. studies (Fig. 7) that all three UBA-domains of NUB1L were required for the binding of FAT10, whereas the UBL-domain was dispensable for this interaction. We were able to detect a weak interaction of FAT10 and NUB1L∆UBL3 in pull- down studies, suggesting that the combination of the first two UBA domains can mediate a weak interaction, but since this interaction was not confirmed in co- immunoprecipitation studies in vivo, the significance of this interaction remains uncertain. Interestingly, the deletion of the 14 amino acids occuring in the natural splice variant NUB1 was insufficient to abolish the binding to FAT10. While studying the interaction of isolated UBA domains with polyubiquitin chains, Raasi et al. divided the UBA-domains into four different groups, de- pending on their ability to discriminate between differently linked polyubiquitin chains and monoubiquitin. The members of the third group – which included all three UBA domains of NUB1L - were not able to interact with ubiquitin at all and may hence be in charge of recognizing ubiquitin-like proteins instead (Raasi et al., 2005). While most reports about UBL-UBA domain proteins focus on ubiquitin or polyubiquitylated substrates, very little is known about their interaction with ubiquitin-like modifiers. In the three first reports on this issue, NUB1 and NUB1L were found to bind to the ubiquitin-like modifier NEDD8 and to acceler- ate its degradation (Kamitani et al., 2001; Kito et al., 2001; Tanaka et al., 2003). Unexpectedly, the interaction of NUB1 and NEDD8 was not mediated by the three UBA domains but rather by a short C-terminal domain. Only the second UBA domain of NUB1L appeared to interact weakly with NEDD8 but it was not required to promote NEDD8 degradation (Tanaka et al., 2003). Subsequently, we reported the robust non-covalent interaction between FAT10 and NUB1L, but in the same series of experiments we failed to detect an interaction of NEDD8 and NUB1L (Hipp et al., 2004). In this study we found that the concerted action of three UBA 53
Chapter 1 domains of NUB1L is required for the binding of FAT10, a ubiquitin-like protein containing two UBL domains. In order to determine if only one or both of the UBL domains of FAT10 are required for binding NUB1L, co-immunoprecipitation studies were performed. These experiments revealed that only the N-terminal but not the C-terminal UBL-domain of FAT10 interacted with NUB1L (Fig. 11). The residues L8, I44, and V70 of ubiquitin, known to be important for the interaction of ubiquitin with other UBA domains, (Vijay-Kumar et al., 1987; Beal et al., 1996; Sloper-Mould et al., 2001; Raasi et al., 2004), are only partially conserved in FAT10. The N-terminal UBL-domain of FAT10 contains the L8 residue and bears a leucine in position 44, but a threonine in position 70. The C-terminal domain, in con- trast, shows in the corresponding residues G8, T44, and A70. While this seems to be in good agreement with our results, it can not be excluded that different residues might be responsible for the interaction between NUB1L and ubiquitin- like modifiers, especially since it could be shown that NUB1L does not bind a ubiquitin-GFP fusion protein (Hipp et al., 2004) or polyubiquitin-chains (Raasi et al., 2005). While interaction of monoubiquitin to UBA domain proteins has been reported (Wilkinson et al., 2001), usually polyubiquitylated proteins are better binders of UBL-UBA domain proteins. This can be rationalized by data on the binding affinity between ubiquitin and the UBL-UBA protein Rad23, where the strength of binding increased exponentially when the chain length of ubiquitin was extended from 1 to 6 units (Raasi et al., 2004). We take our result that one of the two FAT10 UBL domains is sufficient for mediating the interaction with NUB1L (Fig. 11) as further support for the specificity of this interaction. Our data suggest that this interaction is not merely a result of an increase in avidity by connecting two ubiquitin-like domains and thus mimicking a short polyubiquitin chain. Never- theless, since the binding assays performed in our study are of qualitative rather than quantitative nature, we can not rule out that FAT10 or FAT10-GFP bind with a higher affinity than FAT10-N-GFP. Several reports about the influence of UBL-UBA proteins on degradation have already appeared. The deletion of these proteins lead to an inhibition of degradation and accumulation of high molecular weight ubiquitin conjugates (Wilkinson et al., 2001; Chen and Madura, 2002; Funakoshi et al., 2002; Rao and Sastry, 2002; Saeki et al., 2002a). Conversely, overexpression of Rad23 or Dsk2 lead to inhibition of substrate turnover by the 26S proteasome (Kleijnen et al., 2000; 54
Page 1 and 2:
Role of the ubiquitin-like modifier
Page 3 and 4: Acknowledgements . . . . . . . . .
Page 5 and 6: Summary / Zusammenfassung English F
Page 7 and 8: Summary In conclusion, these result
Page 9 and 10: Summary nicht von der katalytischen
Page 11 and 12: The Ubiquitin-Proteasome-System The
Page 13 and 14: Introduction Figure 1: The 26S prot
Page 15 and 16: Introduction are the major source o
Page 17 and 18: Introduction majority of ubiquitin-
Page 19 and 20: Introduction example contained in t
Page 21 and 22: Introduction two modifiers apart fr
Page 23 and 24: Introduction Figure 4: Comparison o
Page 25 and 26: Introduction dependent apoptosis in
Page 27 and 28: Introduction Interestingly, a recen
Page 29 and 30: The Autophagy-Lysosome System Intro
Page 31 and 32: Introduction Figure 5: Molecular ma
Page 33 and 34: Introduction The transport of misfo
Page 35 and 36: Introduction where the proteasome i
Page 37 and 38: Chapter 1 The UBA domains of NUB1L
Page 39 and 40: Introduction Chapter 1 Ubiquitin, a
Page 41 and 42: Chapter 1 no correlation between th
Page 43 and 44: Chapter 1 covered proteins were sep
Page 45 and 46: Chapter 1 Figure 8: Accelerated deg
Page 47 and 48: Chapter 1 Treatment with IFN-γ and
Page 49 and 50: Chapter 1 ing SUMO-1-GFP or the C-t
Page 51 and 52: Chapter 1 Figure 12: Accelerated de
Page 53: Chapter 1 Figure 13: Co-immunopreci
Page 57 and 58: Chapter 1 Rad23 (Verma et al., 2004
Page 59 and 60: Chapter 1 FAT10-GFP expressing vect
Page 61 and 62: Chapter 2 Degradation of FAT10 by t
Page 63 and 64: Introduction Chapter 2 The proteaso
Page 65 and 66: Chapter 2 From our previous data it
Page 67 and 68: Chapter 2 Figure 14: Purified compo
Page 69 and 70: Chapter 2 evaluation of the NUB1L b
Page 71 and 72: Chapter 2 teins which contain intri
Page 73 and 74: Chapter 2 Combined with our studies
Page 75 and 76: Chapter 2 Recombinant expression of
Page 77 and 78: Chapter 2 et al., 2003), both at a
Page 79 and 80: Abstract Chapter 3 During misfolded
Page 81 and 82: Chapter 3 tion between the UPS and
Page 83 and 84: Chapter 3 Figure 18: HDAC6 interact
Page 85 and 86: Chapter 3 Figure 19: Both the BUZ d
Page 87 and 88: Chapter 3 of HDAC6 was required for
Page 89 and 90: Chapter 3 precipitates with HDAC6 (
Page 91 and 92: 90 Chapter 3
Page 93 and 94: Chapter 3 Figure 23: The model conj
Page 95 and 96: Chapter 3 HDAC6 is required for the
Page 97 and 98: Chapter 3 Figure 26: Both ubiquitin
Page 99 and 100: Chapter 3 also revealed endogenous,
Page 101 and 102: Chapter 3 and/or its conjugates is
Page 103 and 104: Chapter 3 (Hipp et al., 2005), pEGF
Page 105 and 106:
Chapter 4 FAT10-deficient mice disp
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Chapter 4 Once the infection is cle
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Chapter 4 As both wild-type as well
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Chapter 4 Peptides. The synthetic p
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Introduction Chapter 5 Antibodies a
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Chapter 5 binant human and mouse GS
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Chapter 5 Figure 34: The antibodies
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Chapter 5 Figure 36: The antibody s
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Discussion 30 years ago, ubiquitin
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Discussion monomeric ubiquitin as w
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Discussion Alternatively, FAT10 cou
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References D. T. Akey, X. Zhu, M. D
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References Y.-H. Chiu, Q. Sun, and
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References S. Gunawardena, L.-S. He
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References J. A. Johnston, M. E. Il
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References F. Lévy, N. Johnsson, T
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References J.-H. Park, H.-S. Jong,
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References P. Schreiner, X. Chen, K
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References S. Vijay-Kumar, C. E. Bu
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Abbreviations aa amino acid AAA ATP
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Amino Acids Alanine Ala A Arginine
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